Molecular epidemiology and collaboration of siderophore-based iron acquisition with surface adhesion in hypervirulent Pseudomonas aeruginosa isolates from wound infections

Iron/siderophore uptake may play an important role in the biofilm formation and secretion of extracellular proteins in Pseudomonas aeruginosa isolates. In the present study, the role of siderophores, heme, and iron regulatory genes in the virulence of Pseudomonas aeruginosa isolates collected from wound infection was investigated. Three hundred eighty-four (384) swab samples were collected from wound infection and identified by phenotypic methods. The quantitative real-time PCR (qRT-PCR) method was evaluated for the gene expressions study. Multi-locus sequence typing (MLST) was used to screen unique sequence types (ST) and clonal complexes (CC). Fifty-five (55) P. aeruginosa isolates were detected in all swab samples. Also, 38 (69.1%) isolates formed biofilm. The prevalence of virulence factor genes was as follows: plcN (67.2%), exoY (70.9%), exoA (60.0%), phzM (58.1%), plcH (50.9%), lasB (36.3%), aprA (69.1%), lasA (34.5%), nanI (74.5%), exoU (70.9%), exoS (60.0%), exoT (63.6%) and algD (65.4%). According to qRT-PCR, genes regulating iron uptake were highly expressed in the toxigenic isolate. The highest expressions levels were observed for hemO, hasR, and pvdA genes in the biofilm-forming isolates. The MLST data confirmed a high prevalence of ST1, ST111, and ST235, with six, five, and 12 clusters, respectively. ST235 and ST1 were the most present among the biofilm-forming and toxigenic strains. Also, the nuoD gene with 54 and guaA with 19 showed the highest and lowest number of unique alleles. We demonstrated that iron/siderophore uptake is sufficient for biofilm formation and an increase in the pathogenesis of P. aeruginosa. These results suggest that the iron/siderophore uptake system may alter the MLST types of P. aeruginosa and predispose to bacterial pathogenesis in wound infections.

The frequency of biofilm-forming isolates. The prevalence of biofilm-forming P. aeruginosa is shown in Table 1 and Fig. 2. Out of the 55 isolates, 28 (50.9%) isolates were found to form strong biofilms, ten isolates (18.1%) moderately formed biofilm, and 19 isolates (34.5%) were biofilm non-producers.
The frequency of VF genes. Out of 55 isolates of P. aeruginosa, 9 (15.7%) isolates carry all VFs genes.

Molecular analysis of siderophores, heme, and iron regulatory genes.
The results of the expression levels of iron-uptake and siderophore regulatory genes are shown in Fig. 2. Overexpression of iron/siderophore regulatory genes in MDR and XDR strains was observed. Moreover, iron-uptake regulatory genes are less than the expression levels in P. aeruginosa ATCC27853. Moreover, hemO, hasR, and pvdA genes are highly expressed in the biofilm-forming isolates. In isolate 60H, iron-uptake and siderophore regulatory genes showed higher expression levels. Whereas, in PA49 and PA221, iron-uptake regulatory genes showed down-expression compared to PA31 and PA60H isolates.
Analysis of hit map tree of gene expression. Figure 2 shows a comparison among the expression levels for the iron-uptake and siderophore regulatory genes in all 55 isolates of P. aeruginosa. The pchA, pvdA, and fptA genes had different activities among the clinical isolates. However, these genes showed a slight expression level in some biofilm-forming isolates and a high expression level in some antibiotic-sensitive strains. Siderophore regulatory genes are expressed to a great extent in strong biofilm-forming isolates. These genes were also highly expressed in colistin-intermediate strains. The majority of the iron-uptake regulatory genes were expressed at deficient levels in the non-toxigenic strains of P. aeruginosa. Also, siderophore regulatory genes overexpressed in biofilm-forming and toxigenic isolates. The heat map tree shows a significant relationship between antibiotic resistance and the activity of iron-uptake regulatory genes. www.nature.com/scientificreports/ Analysis of MLST dendrogram phylogenic tree. As shown in Fig. 3, the neighbor-joining tree based on nucleotide difference in sequence data of each housekeeping gene was constructed. Sequencing of housekeeping genes of all 55 representative isolates of P. aeruginosa showed 28 sequence types and 28 clusters among all isolates tested. However, ST1, ST235, and ST111 were the most common STs among P. aeruginosa isolates. These three STs also showed the highest frequency among MDR and XDR strains. The NBJ tree of the ST235 gene included eight isolates (PA32, PA60H, 97Z, 101Z, 33H, 57Z, 33C, 14H, and 11H) into one significant cluster. Nineteen unique alleles were identified for each housekeeping gene: 19 for the guaA gene, 54 for the nuoD gene, 39 for aroE gene, 33 for the trpE gene, 41 for the ppsA gene, and 50 for the guaA gene. The GC % observed in seven housekeeping genes ranges from 35 to 51%. The sequence types found were ST1, ST10, ST63, ST60, ST125, ST197, ST216, ST29, ST147, ST511, ST70, ST101, ST486, and ST509. ST15, ST111, ST29, ST 147, ST70, and ST101 were among the novel STs from the west of Iran.
Relationship between virulence factors and iron uptake. The results of the statical analysis are shown in Table 1 and Fig. 4. The virulence profiles are significantly associated with biofilm formation (p ≤ 0.05). No association between the VFs and colistin, amikacin, and piperacillin/tazobactam resistance was detected. A strong correlation was observed among virulence profiles and expressions of siderophores, heme, and iron regulatory genes (p ≤ 0.001).

Discussion
In the current study, 94.5% of P. aeruginosa were sensitive to colistin, and 67.2% of isolates were resistant to ceftazidime. The observations also agree with the results reported by Nasser et al. 19 . However, some researchers from Nigeria 20 reported a high frequency of colistin-resistant P. aeruginosa strains. The findings of the current study do not support the previous research. However, the mechanisms through which these Gram-negative bacteria acquire polymyxin resistance remain poorly known. Based on these comparisons, the recent appearance www.nature.com/scientificreports/ of polymyxin resistance highlights the critical need of gaining a better knowledge of the link between various resistance mechanisms and virulence in these Gram-negative bacteria 20 .
In the current study, 66.6% of isolates were able to form a biofilm. These results reflect those of Asati et al. and Kamali et al., who also found that biofilm formation occurs significantly more in burn infections 11,14 .
Based on biofilm results, the expression levels of siderophores, heme, and ferric citrate iron genes on strong biofilm-producing strains were higher than in moderate biofilm producers. There was also a strong correlation between biofilm formation and high expression of siderophores and heme genes. Zhang et al. and Abbas et al. stated that the overexpression of the tonB gene in the biofilm-forming strains compared to the non-biofilmforming strains 7,8 . Poole et al., found that the tonB gene plays an essential role in siderophore-mediated iron uptake 23 . In line with the mentioned studies, the fold change of the tonB gene in isolates without biofilm (PA49, PA50Z, PA42Z, PA56, PA221, and PA70H) showed the lowest level.
Our results showed the down-regulation of siderophores and heme genes in non-biofilm-forming isolates (PA42Z, PA49, PA221, and PA70H). However, many genes are involved in siderophores, heme, and ferric citrate iron absorption in P. aeruginosa. Thus, the bacterium is trying to form a biofilm to preserve its survival against Table 1. Frequency of antibiotic resistance, and virulence factors in biofilm producing and non-biofilm producing P. aeruginosa wound collection. aeruginosa. Isolates with the potential to form biofilm, non-biofilm-forming, versus toxigenic and non-toxigenic strains. (a) Heatmap iron-uptake and siderophore system expression patterns in all 55 P. aeruginosa. Blue represents up-regulation and red down-regulation relative to control. (b) The volcano plots of iron-uptake and siderophore system genes expressions between toxigenic and non-toxigenic isolates. Expressions analysis based on p-value and log 2 (fold-change) at an α level of 0.05 and. (c) The volcano plots of iron-uptake and siderophore system genes expressions between biofilm and non-biofilm forming isolates, discriminated based on p-value and log 2 (fold-change) at an α level of 0.05. (d) Expression levels of iron-uptake and siderophore system genes between toxigenic a non-toxigenic isolate. Expressions analysis based on p-value and log 2 (fold-change) at an α level of 0.05. (e): Expression levels of iron-uptake and siderophore system genes between biofilm non-biofilm forming isolates, discriminated based on p-value and log 2 (fold-change) at an α level of 0.05. Error bars standard errors: 0.05. Student's t-test and Tow-Way ANOVA test were performed for testing differences between groups. *p < 0.05, **p < 0.001, ***p < 0.0001.  24,25 . Moreover, Singh studied the effects of iron on biofilm formation and found that, in the absence of lactoferrin, if no functional iron uptake system were present, biofilms would still form flat, thin colonies 26 .

Scientific Reports
In the present study, we found that more than 50% of P. aeruginosa isolates carried one or more VF genes. Also, VF genes were more abundant in biofilm-forming strains compared to non-biofilm-forming strains. Further, some isolates carrying all or many VF genes (PA11, PA11H, PA101Z, PA60H, PA33C, PA32, and PA31) showed high expression levels of tonB, phuR, hemO, hasR, and pvdA genes. Some researchers from the USA 27 , Cyprus 15 , and China 28 also confirmed the association between biofilm formation and increased iron/siderophore regulatory genes. They also showed that P. aeruginosa requires iron to form cofactors of enzymes that play essential roles in electron transfer and other critical cellular processes. Iron also signals biofilm formation, but the level of iron necessary to promote biofilm formation exceeds that required for assimilatory purposes. Besides, the production of several virulence determinants, such as exotoxin A, is regulated in response to iron 29 .
Based on the present study, tonB, pchA, and pvdA had a variable expression in toxigenic and non-toxigenic isolates. Also, comparing the value for pchA and fptA in susceptible isolates to antibiotics with the corresponding value in the resistant isolates, it was observed that the value in the susceptible isolates was consistently lower than www.nature.com/scientificreports/ the resistant isolates. In non-toxigenic and antibiotic-sensitive strains, biofilm formation may play a direct role in iron uptake and the activity of siderophore controller genes. However, in line with the ideas of Klebba et al., it can be concluded that the tonB complex consists of the inner membrane protein tonB, which spans the periplasmic space and associates with outer membrane iron receptors 30 . Nonetheless, some researchers from Canada 17 , Germany 31,32 , and Switzerland 18 proved that P. aeruginosa also contains two heme uptake systems whose outer membrane receptors are HasR and PhuR energized PhuUV ABC transporter tonB complex, respectively. MLST is considered the "gold standard" of typing for many bacterium species. Based on our MLST results, the data generated by nucleotide sequence analysis are unambiguous and easily transferable, and comparable between laboratories, and the methodologies used are both generic and highly reproducible. This method was developed as a comprehensive typing approach that addressed repeatability, reliability, affordability, and throughput requirements 28 . A significant benefit of MLST analysis is that it generates clear sequencing data that are suited for population structure and epidemiological investigation 26,27 .
According to our observation in wound infections, dangerous STs like ST11, ST1, and ST235 had high expression levels of siderophores, heme, and ferric citrate iron genes. These STs have been frequently reported in wound infections, and high levels of antibiotic resistance have also been seen in this ST. In a study, Kim et al. 33 found that wound infection isolates of P. aeruginosa were highly pathogenic and extremely damaged skin tissue. They also showed the overexpression of siderophores and heme genes in isolates of wound infections. ST11 and ST235 were identified as dangerous strains in wound infections in studies by Koutsogiannou et al. 34 and Omar et al. 35 .
In this study, among 55 isolates, 28 sequence types were detected, and ST1, ST235, and ST111 were the most common STs in P. aeruginosa isolates. Moreover, the remaining 12 isolates were found to belong to ST235. These data are in good accordance with what Guzvinec et al. 36 reported for a collection of P. aeruginosa isolates, showing that ST11 and ST253 were more common STs in P. aeruginosa isolates. Although most of the ST253 and ST1 in this study were among the most founded STs worldwide, none of these isolates have the same VF profile, and most of them are considered a toxigenic strain. Moreover, ST3401, ST2088, ST2088, ST1078, and ST926 considered non-toxigenic strains. Our current findings are consistent with other studies from Iran 37 , Brazil 38 , and France 39 , where they found ST111, ST235, and ST233 as predominant STs from biofilm-forming P. aeruginosa isolates.
However, ST1, ST235, and ST11 also showed the highest frequency among the MDR and XDR strains. The NBJ tree of the ST235 gene included eight isolates (PA32, PA32, PA60H, 97Z, 11H, 101Z, 33H, 57Z, 33C, 14H, 11Z, and 11H) into one central cluster. Interestingly, some sequence types were among the novel STs from the west of Iran. Similar to our results, some STs and variants were seen among resistant and virulent isolates elsewhere by Annear et al. 40 and Liu et al. 41 ; they also identified ST1, ST235 mostly found in hyper pathogenic isolates. www.nature.com/scientificreports/ Our data analysis indicated a significant correlation between VF genes and the expression of siderophores, heme, and ferric citrate iron genes (p ≤ 0.001). However, the VF gene profiles are significantly associated with biofilm formation (p ≤ 0.05). Similarly, VF genes were more prevalent in biofilm-forming strains. However, there are no accurate reports of the frequency of sequence typing of P. aeruginosa isolates in Iran. Like our results, there have been various reports of those associations in Italy 13 and Belgium 42 .
In conclusion, our knowledge of this study showed a strong association between regulation genes of siderophores, heme, and ferric citrate iron uptake and biofilm formation. Also, the uptake pathway of siderophore/iron in P. aeruginosa plays a significant role in bacterial pathogenesis. Therefore, in chronic wound infections, biofilm formation by siderophores, heme, and ferric citrate iron uptake occurs in P. aeruginosa. However, discovering a new clonal complex in a wound infection of P. aeruginosa without the previously recognized predisposing factors for the emergence of such strains; has led to a revision of our understanding of wound infection clonal complex epidemiology. Finally, molecular typing can simplify identifying appropriate infection control measures to lower the mortality and morbidity of wound infections.

Materials and methods
Ethics statement. The study was approved by the institutional review boards from the Ethics Committee of Hamadan University of Medical (No: IR.UMSHA.REC.1398.481), which allowed the phenotypic and genotypic characterizations of the Hypervirulent P. aeruginosa isolates. The project was not involved in the collection and analysis of the demographics and clinical information of any patient. Informed written consent was obtained from the study population or their guardians after providing a full explanation of the study.

Study design and collection of isolates.
Our study was conducted at Hamadan, Iran Hospitals (Farshchian (Sina) Hospital, Be'sat Hospital, and Beheshti Hospital) with about 1000 beds, serving a population in a large metropolitan region. The Hamadan Hospitals and the Hamadan microbiology department have a high reputation in town, ranking first in various medical indicators.
The study included patients with complicated wound infection symptoms (chronic or acute wounds), in Burn Departments of our center who had received carbapenems and other antibiotics before admission or during their hospital stay. Inclusion criteria were treated with antibiotics for at least 24 h and the presence of positive microbiological cultures. Exclusion criteria were negative microbiological cultures or no culture request and multiple or unknown origins of the infection. In Microsoft Office Excel 2019 (Microsoft Corporation, Redmond, WA, USA), stratified sampling was utilized to extract isolates from each layer, and random sampling was employed to acquire a total of burn wound sample isolates.
In the present study, 55 P. aeruginosa were isolated from different burn wound samples (384 samples) between July 2019 and April 2020 at Hamadan Hospitals. Burn wound swabs were collected in aseptic conditions, and samples were transported to the microbiology department. Swabs were immediately inoculated on MacConkey Agar (Hi-Media, India). Organisms were identified using Cetrimide Agar (Hi-Media, India) and standard biochemical tests. Finally, all isolates were stored at − 80 °C in Luria-Bertani (Sigma Aldrich) containing 10% glycerol (W/V) (Sigma Aldrich). All methods which used in this study were carried out following relevant guidelines and regulations which were approved by the microbiology department, Hamadan university of medical sciences.
Antimicrobial susceptibility testing (AST). The AST was performed using the disk diffusion method (DDM) based on the Clinical & Laboratory Standards Institute (CLSI) guidelines version 2020 for all P. aeruginosa isolates 38 . The Liofilchem® MIC Test Strips (Liofilchem, Italy) was used to determine the minimum inhibitory concentration to identify colistin-resistant strains. P. aeruginosa ATCC 15442 and P. aeruginosa ATCC 27853 were used as control strains 43 .
Phenotypic screening of biofilm-forming strains. The crystal violet method (CVM) for the screening of biofilm-forming strains was done according to the Manandhar et al. study 44 . The optical density (O.D.) of each well was measured at 570 nm using an ELISA reader (BioTek Instruments, Inc, USA). As the bacteria form biofilm and adhere to the wells, these OD values were taken as bacterial adherence index. The standard strain Staphylococcus epidermidis ATCC 35984 was used as a control for biofilm production. The standard strain Staphylococcus epidermidis ATCC 12228 was used as the non-biofilm producer control strain.
DNA extraction and detection of VF genes. The boiling method was used for DNA extraction according to Dehbashi et al. study 2 . Nanodrop (Hangzhou Allsheng Instruments Co., Ltd, China) was used to measure DNA concentration. For screening, the VF genes were performed using the specific primers listed in Table 2 were used. The programmable thermal cycler (Eppendorf, Germany) PCR device was applied in all PCR reactions. The 25 μl reaction mixture contained 12.5 µl of master mix (Ready Mix TM-Taq PCR Reaction Mix, Ampliqon, Denmark), 0.5 μM concentration of each primer, one μl of the 5 ng/μl genomic DNA template, and 11.5 µL of molecular biology grade water. In each round of amplification, sterile water was used as a negative control.
RNA extraction, synthesis of cDNA, and quantitative real-time PCR (qRT-PCR). Total RNA extraction and synthesis of cDNA were performed according to Dehbashi et al. study 37 . PCR reactions were performed in 96-well microplates (ABI-Step One-Plus) using the ABI-Step One-Plus Real-time System, ABI, USA. qRT-PCR was carried out using 4 μl of 2 × FIREPol Master Mix (Solis BioDyne, Tartu, Estonia), 0.5 μl (10 pM) forward and reverse primers were 2 μl template cDNA, and 13 μl RNase free water to a final volume of 20 μl. The PCR protocol was designed for 40 cycles, and a melting-curve analysis (65-95 °C, fluorescence read www.nature.com/scientificreports/ value ± SE. As recommended by the manufacturer, all baseline and threshold values were reviewed and manually adjusted as required. Further analysis was performed in Data-Assist (Applied Biosystems, CA, USA). The p-value was calculated based on a two-sample, two-tailed Student's t-test for the calculated Fold change (relative to the epilepsy group). A dendrogram was constructed from a distance-based matrix of the allelic profiles using the neighbor-joining method. In this study MEGA version 6, the tree-drawing tools PHYLIP (available at http: //pubmlst.org/analysis) and the iTOL online tool (http:// itol. embl .de/itol.cgi) were used.
The phenotypic antibiotic susceptibility profiles and the presence of genes were converted to binary code. For each antibiotic, 0 indicated susceptibility, and one (1) indicated resistance; likewise, a gene's presence was designated as one (1)